Antigen and method for production thereof

ABSTRACT

The invention refers to a method for producing an antigen comprising at least one hydrophobic or partially hydrophobic antigen molecule from a virus, a  bacterium,  fungus, protozoan, parasite, a human neoplastic cell or an animal neoplastic, tumour or 5 cancer cell, the method comprising the steps of providing a virus, or cell comprising an antigen molecule, purifying the cell comprising the antigen molecule, solubilizing the antigen molecule in a solubilizing agent that preserves an intact antigen molecule upon solubilisation and reconstituting the antigen molecule in a lipid-binding polypeptide that provides a lipid membrane mimicking environment and a reconstituted antigen particle 10 obtained by this method.

TECHNICAL FIELD

The invention relates to an antigen and method for production thereof,in particular to a reconstituted antigen particle comprising at leastone hydrophobic or partially hydrophobic antigen molecule from a virus,a bacterium, fungus, protozoan, parasite, a human neoplastic cell or ananimal neoplastic, tumour or cancer cell, suitable for producing anantigenic composition or a vaccine for protecting against membraneenveloped viruses or bacteria, and is in particular suitable forproducing an HIV vaccine such as Human Immunodeficiency Virus Type 1(HIV-1) vaccine, but is also useful for producing vaccines or tools forproducing vaccines for other membrane enveloped viruses or bacteria.

TECHNICAL BACKGROUND

The HIV-1/AIDS pandemic is the foremost infectious disease causing deathand morbidity worldwide. The pandemic, which affects primarily youngadults between 15 and 40 years of age, is a socio-economic as well as aburning health issue. In the countries of sub-Saharan Africa worstaffected by the pandemic, the loss of young adults has impacted upon theeconomic output from these countries.

An effective approach to curbing the future impact of this viral diseaseis through the development and delivery worldwide of a safe, effectiveand affordable vaccine that prevents infection and transmission of thevirus.

Also alternative medical treatments to a vaccine exist today. EfficientHIV-1 drugs exist today, but a downside is the immense costs associatedwith treatment making it less available for underprivileged third worldcountries and an economic burden for wealthier countries. Anotherdownside of current treatments is the development of drug resistance.Consequently the drugs are only life prolongers and not an absolutecure. The best strategy to prevent an HIV-1 pandemic, resulting in AIDSpatients, would most likely be an effective vaccine. This has been ahighly prioritized research field for over 30 years but there is stillno working vaccine in sight.

Typically, since a natural defense mechanism of humans and animalsagainst viral diseases is based on a production of protectiveantibodies, a primary function of many vaccines against such diseaseshas been to elicit protective antibodies against infectious agents orparts thereof.

Since the discovery of HIV-1 in 1984, efforts to develop a prophylacticvaccine against HIV-1/AIDS infection have been intense and it is nowclear that this is an unprecedented vaccine challenge. Due to theextreme genetic variability of HIV-1, a successful vaccine would need toelicit high-titer neutralizing antibodies not only against one or a fewvirus strains but against the enormous breadth of genetic diversity thatexists among circulating viral variants, so-called broadly neutralizingantibodies (bNAbs). This is only possible if conserved and exposed viraldeterminants are targeted by the immune response. A further challengewith HIV-1 is that the envelope glycoprotein (Env) spikes are unstablecomplexes and vaccine candidates where stable spikes that preserve, orsufficiently well mimic, the native conformation of the full-lengthfunctional HIV-1 spike were so far not developed. This is likely to be amajor reason for the failure of most HIV vaccine candidates developed sofar.

Although the vaccine field waits for a breakthrough a lot of informationhas recently been gained about broadly neutralizing antibodies (bnAb).BnAb are antibodies that have been found in some HIV-1 infected personsand they are of high interest because they, binds to and, prevent mostviral strains to enter target cells. These results have shown itpossible for an individual's immune system to evolve a working antibodyresponse towards HIV-1. But, still no vaccine approach has been able togenerate bnAb that targets the HIV-1 virus in humans.

DESCRIPTION OF THE INVENTION

It has been realized that the targets for HIV-1 neutralizing antibodiesare the exterior envelope glycoprotein, gp120, and the trans membraneglycoprotein, gp41 that are derived from proteolytic cleavage of thegp160 precursor protein in the Golgi apparatus of virus-producing cells.Trimers of non-covalently associated gp120 and gp41 form the functionalspike complexes, which mediate viral entry into host target cells. Hostselection pressures have made most circulating HIV-1 isolates extremelyresistant to neutralizing Abs and thus only very few conserved epitopesare exposed on the functional virus spike.

These conserved targets have been subject to intense interest astemplates for HIV-1 immunogenic design and include the highly conservedCD4 binding site (CD4bs) on gp120, the membrane-proximal region of gp41,a cluster of glycans on the gp120 outer domain and an epitope proximalto the base of the variable region 1 and 2 that appears to betrimer-specific. These antibody specificities were all defined by serummapping studies of HIV-1 infected individuals possessing bNAb activityand the corresponding monoclonal antibodies (MAbs) were isolated andconfirmed to mediate extremely potent and broad HIV-1 neutralization.These studies provide hope for the development of a vaccine againstHIV-1 as if such Abs were induced by Env immunization they would providea first line of defence against HIV-1 exposure and blunt the acuteviremia resulting in a lower viral load “set-point”. This would lead toan improved clinical prognosis for the affected individual and areduction in transmission rate within the human population, twoconsiderable achievements for a vaccine.

Many current vaccines against microbial pathogens comprise liveattenuated or non-virulent strains of the causative microorganisms. Manyvaccines comprise killed or otherwise inactivated microorganisms. Othervaccines utilize purified components of pathogen lysates, such assurface carbohydrates or recombinant pathogen-derived proteins. Vaccinesthat utilize live attenuated or inactivated pathogens typically yield avigorous immune response, but their use has limitations. For example,live vaccine strains can sometimes cause infectious pathologies,especially when administered to immune-compromised recipients. Moreover,many pathogens, particularly viruses, undergo continuous rapid mutationsin their genome, which allow them to escape immune responses toantigenically distinct vaccine strains.

Given the difficulty of vaccine development, many vaccines are inextremely short supply. For example, as of October 2007, there areinfluenza, varicella, and hepatitis A vaccine shortages in the UnitedStates. In some instances, vaccine shortages occur because not enoughmanufacturers devote their facilities to vaccine production to keep upwith demand. In some cases, vaccine shortages are attributed to lowpotency of the vaccine, which means a large amount of vaccine productmust be administered to each individual in order to achieve aprophylactic effect. For example, some vaccines cannot be administeredas an intact organism (even if attenuated or killed) because they causeinfectious pathologies. Instead, such vaccines usually comprise purifiedpathogen components, which typically lead to a much less potent immuneresponse.

SUMMARY OF THE INVENTION

Thus, there is a need in the art for systems and methods for producinghighly immunogenic, effective vaccines. There is also a need forimproved vaccine compositions that can potently induce long-lastingimmune responses. For the treatment and prevention of infectiousdiseases, there is a need for improved vaccine compositions that arehighly immunogenic but do not cause disease.

This problem is solved by a method for producing an antigen comprisingat least one hydrophobic or partially hydrophobic antigen molecule froma virus, a bacterium, fungus, protozoan, parasite, a human neoplasticcell or an animal neoplastic, tumour or cancer cell, the methodcomprising the steps of

-   -   providing a virus, or cell comprising an antigen molecule,    -   purifying the cell comprising the antigen molecule,    -   solubilizing the antigen molecule in a solubilizing agent that        preserves intact an antigen molecule upon solubilisation,    -   reconstituting the antigen molecule in a lipid-binding        polypeptide that provides a lipid membrane mimicking        environment.

Surprisingly, it has been found that a lipid-binding polypeptide(protein), preferably a saposin-like protein, makes it possible toreconstitute a solubilized antigen from a virus, a bacterium, fungus,protozoan, parasite, a human neoplastic cell or an animal neoplastic,tumour or cancer cell. In particular the saposin-like protein acts as alipid membrane mimicking environment that allows to produce stableantigen molecules such as membrane proteins, or membrane proteinparticles, having preserved structure and functionality, also atelevated temperatures of about 37° C. by means of the method accordingto the present invention.

Preferably the lipid-binding polypeptide is a protein of the saposinfamily, namely at least one of saposins A to D. Further a mixture of twoor more of these particles can be used for the purposes of theinvention. Particles composed of or obtained from the lipid-bindingpolypeptide and at least one hydrophobic or partially hydrophobicantigen molecule subsequently are referred to as lipid bindingpolypeptide/antigen particles.

Unexpectedly, it was found that in the presence of a lipid-bindingpolypeptide, preferably a saposin-like protein, a solubilized antigenself-assembles into stable lipid binding polypeptide/antigen particleswithout the need of a laborious upstream liposome preparation step.

The lipid-binding polypeptides used in the invention are capable ofincorporating or harboring a variety of hydrophobic compounds such aslipids and membrane proteins at a physiological pH, giving rise tonanoscale complexes that are soluble and stable in an aqueousenvironment.

The lipid binding polypeptide preferably is a saposin-like protein(SAPLIP) or a derivative or truncated form thereof. Due to the highdegree of structural and functional conservation among SAPLIPs, thefeatures and advantages of certain embodiments of the invention withsaposin A as lipid binding polypeptide are expected to also apply toother embodiments using other SAPLIPs or derivatives or truncated formsthereof as lipid binding polypeptide of the invention.

According to a preferred embodiment, the SAPLIP is saposin A, B, C or D,in particular a saposin selected from (Homo sapiens, Equus caballus, Bostaurus, Mus musculus, Oryctolagus cuniculus, Rattus norvegicus orXenopus laevis) saposin A, saposin B, saposin C or saposin D. In oneembodiment, the SAPLIP is (Homo sapiens, Equus caballus, Bos taurus, Musmusculus, Oryctolagus cuniculus, Rattus norvegicus or Xenopus laevis)saposin A, saposin B or saposin D.

Saposin C is special among the saposins in that it is capable ofinducing membrane fusion, a feature which is not exhibited by the othersaposins. The membrane fusion activity may not always be desirable.According to a particular embodiment of the invention the lipid bindingpolypeptide is a saposin-like protein (SAPLIP) or a derivative ortruncated form thereof, provided that the SAPLIP is not saposin C orprovided that the SAPLIP is not saposin C or a derivative or truncatedform thereof.

In one embodiment, the SAPLIP is of human origin (i.e. a Homo sapiensSAPLIP).

In a preferred embodiment, the SAPLIP is saposin A, preferably (Homosapiens, Equus caballus, Bos taurus, Mus musculus, Oryctolaguscuniculus, Rattus norvegicus or Xenopus laevis) saposin A, andparticularly preferred human saposin A, the amino acid sequence of whichis given as SEQ ID NO. 1. Saposin A is a known protein. Its expression,purification and crystallization as LDAO-detergent complex is forexample, described in PNAS, Vol. 109, No. 8 (2012) 2908-2912 (Popovic etal.).

When a derivative or truncated form of saposin A is used aslipid-binding polypeptide used according to the invention, saidderivative or truncated form should be amphipathic, form at least onealpha helix, and be capable of self-assembling together with solubilizedantigens into lipid-binding polypeptide/antigen particles when employedin the preparation process according to the invention which is describedin detail below. As used herein, the term “amphipathic” refers topolypeptides or molecules having both hydrophilic and hydrophobicregions.

Preferably, if a derivative of a SAPLIP is used, the six cysteineresidues corresponding to the six cysteines in the SAPLIP foundingmember saposin A should be present. It is referred in this respect tothe positions of the cysteines in the sequence comparison in FIGS. 4Aand 4B of Bruhn (2005), Biochem J 389 (15): 249-257, which figures arehereby specifically incorporated by reference.

The lipid binding polypeptide according to the invention may alsoinclude one or more non-natural amino acids; amino acid analogs, or apeptidomimetic structure, in which the peptide bond is replaced by astructure more resistant to metabolic degradation. Hence, the product ofthe process of the invention and the product of the invention may be aparticle that consists essentially of the lipid-binding polypeptide anda reconstituted antigen. The lipid-binding polypeptide particles/antigenparticles are robust over concentrating using standard centrifugalfilter units, freezing and thawing. Moreover, practical experimentsrevealed that these particles display a certain degree ofthermostability. In addition, it is possible to freeze-dry, store andre-hydrate the particles without any major quality deteriorationobservable.

By means of reconstituting the antigen molecules in the lipid-bindingpolypeptide, the antigen molecules are solubilized into clusters oflipid binding polypeptide particles without adding any additionallipids.

Such particles can be used in immunogenic compositions, for example, asvaccine components. Antigen proteins of interest include, withoutlimitation, gp160, wherein gp160 corresponds to gp120/gp41, of HumanImmunodeficiency Virus, envelope glycoproteins of Herpes simplex virusor measles virus, the “spike” protein of the SARS virus, hemaglutininligand of influenza virus or parainfluenza virus. Exemplary bacterialantigens, or antigen molecules, include, but are not limited to, cellsurface proteins such as the M6 protein or M proteins of Streptococcuspyogenes, fimbrillin of Porphryomonas gingivalis, InIB or ActA ofListeria monocytogenes, YadA of Yersinia enterocolitica, IcsA ofShigella flexneri, invasin of Yersinia pseudotuberculosis, products ofthe acf gene of Vibrio cholerae, capsular material comprising thepoly-D-glutamate polypeptide of Bacillus anthracis, fibrinogen/fibrinbinding protein of Staphylococcus aureus, V and/or W antigens ofYersinia pestis (especially from a vaccine strain such as EV76) or fromYersinia enterocolytica or Yersinia pseudotuberculosis, and flagellin orporin of Campylobacter jejeuni. Similarly, O antigens of Salmonellatyphi, Salmonella choleraesuis, and Salmonella enteritidis can becombined with lipid-binding polypeptides to form lipid-bindingpolypeptides/antigen particles, using the proteins and methods describedherein.

One aspect of the invention is the provision of vaccines. A vaccineaccording to the invention typically contains an antigen. In oneembodiment of the invention, the antigen is physically ‘bound’ to thelipid-binding polypeptide by covalent or non-covalent means.Non-covalently bound includes, for example, ionic bonding, hydrophobicbonding, physical entrapment, and the like, all described in greaterdetail below. Such nano-carriers which themselves carry an antigen areincluded in the category referred to below as vaccine nano-carriers. Inanother embodiment, the nano-carrier has bound to it animmunostimulatory agent for enhancing, suppressing, directing, orredirecting an immune response, preferably to an antigen. In this case,the antigen may be mixed with the preparation of agent boundnano-carrier to which the immunostimulatory agent is bound form thevaccine. The antigen, of course may also be bound to a nano-carrier,including as discussed below, the same nano-carrier to which theimmunostimulatory agent is bound.

According to another aspect, the present invention further providesimmunogenic compositions comprising lipid binding polypeptide/antigenparticles with at least one hydrophobic or partially hydrophobic antigenmolecule being incorporated in the particles, preferably together with apharmaceutically acceptable carrier. Optionally an adjuvant and/or animmune stimulant, such as a chemokine, can be incorporated into thecomposition. The particles allow the stabilization and solubilisation ofa hydrophobic antigen, with the maintenance of the native conformationof the antigen, and with the presentation of hydrophilic regions of theantigen exposed to the aqueous environment, according to our bestunderstanding leading to an improved immune response in the human oranimal to which the immunogenic composition has been administered.

Antigens which are hydrophobic or partially hydrophobic can beformulated into immunogenic compositions for administration to a humanor animal in which an immune response, either cellular or humoral, aredesired. The incorporation of the antigen into the lipid-bindingpolypeptide/antigen particles with the method according to the inventionallows the preparation of stable aqueous preparations which do not havea tendency to aggregate. Incorporation does not mean that the antigen isfully surrounded by the lipid-binding polypeptide. At least oneantigenic determinant of the antigen is presented to the aqueous phase,with the more hydrophobic portions of the antigen being buried withinthe hydrophobic central region of the lipid binding polypeptide/antigenparticle. The antigen incorporated within the particles can be aprotein, such as a cell membrane protein or another antigen such as aviral envelope protein, or it can be a lipopolysaccharide or alipooligosaccharide.

The antigen can be derived from a particle or cell, such as a virus, forinstance an enveloped virus, a bacterium including, but not limited to,a bacterium, fungus, protozoan, parasite, or it can be derived from aparticular type of tumour or cancer. The antigen-containing particlecomposition can be administered in prophylactic or therapeutic treatmentregimens to generate an immune response, and administration of theseparticles can be carried out in combination with other vaccinepreparations for priming and/or boosting.

Cancers (neoplastic conditions) from which cells can be obtained for useas an antigen source in the methods of the present invention includecarcinomas, sarcomas, leukemias and cancers derived from cells of thenervous system. These include, but are not limited to bone cancers(osteosarcoma), brain cancers, pancreatic cancers, lung cancers such assmall and large cell adenocarcinomas, rhabdosarcoma, mesiothelioma,squamous cell carcinoma, basal cell carcinoma, malignant melanoma, otherskin cancers, bronchoalveolar carcinoma, colon cancers, othergastrointestinal cancers, renal cancers, liver cancers, breast cancers,cancers of the uterus, ovaries or cervix, prostate cancers, lymphomas,myelomas, bladder cancers, cancers of the reticuloendothelial system(RES) such as B or T cell lymphomas, melanoma, and soft tissue cancers.

The terms “neoplastic cell”, “tumour cell”, or “cancer cell”, usedeither in the singular or plural form, refer to cells that haveundergone a malignant transformation that makes them harmful to the hostorganism. Primary cancer cells (that is, cells obtained from near thesite of malignant transformation) can be readily distinguished fromnon-cancerous cells by well-established techniques, particularlyhistological examination. The definition of a cancer cell, as usedherein, includes not only a primary cancer cell, but also any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.When referring to a type of cancer that normally manifests as a solidtumour, a “clinically detectable” tumour is one that is detectable onthe basis of tumour mass; e.g., by such procedures as CAT scan, magneticresonance imaging (MRI), X-ray, ultrasound, or palpation. Biochemical orimmunologic findings alone may be insufficient to meet this definition.

Pathogens to which multiple antigen immunological responses areadvantageous include viral, bacterial, fungal and protozoan pathogens.Viruses to which immunity is desirable include, but are not limited to,hemorrhagic fever viruses (such as Ebola virus), immune deficiencyviruses (such as feline or human immunodeficiency viruses), herpesviruses, coronaviruses, adenoviruses, poxviruses, picornaviruses,orthomyxoviruses, paramyxoviruses, rubella, toga viruses, flaviviruses,bunyaviruses, reoviruses, oncogenic viruses such as retroviruses,pathogenic alphaviruses (such as Chikungunya virus), rhinoviruses,hepatitis viruses (e.g. Group B, C), influenza viruses, among others.Bacterial pathogens to which immune responses are helpful include,without limitation, staphylococci, streptococci, pneumococci,salmonellae, escherichiae, yersiniae, enterococci, clostridia,corynebacteria, hemophilus, neisseriae, bacteroides, francisella,legionella, pasteurellae, brucellae, mycobacteriae, bordetella,spirochetes, actinomycetes, chlamydiae, mycoplasmas, rickettsias, andothers. Pathogenic fungi of interest include but are not limited toCandida, cryptococci, blastomyces, histoplasma, coccidioides,phycomycetes, trichodermas, aspergilli, pneumocystis, and others.Protozoans to which immunity is useful include, without limitation,toxoplasma, plasmodia, schistosomes, amoebae, giardia, babesia,leishmania, and others. Other parasites include the roundworms,hookworms and tapeworms, filiaria and others.

A further aspect of the invention is the administration of theantigen-containing immunogenic particle compositions of the invention toa human or animal (e.g. horse, pig, cow, goat, rabbit, mouse, hamster)to generate immune responses, such as production of antibody specific tothe antigen or a cellular response such that cells or tissues sharingthe antigen are the subject of a cellular or cytotoxic immune response.Sera or cells collected from such humans or animals are useful inproviding polyclonal sera or cells for the production of hybridomas thatgenerate monoclonal sera, such antibody preparations being useful inresearch, diagnostic, and therapeutic applications.

While the generation of an immune response includes at least some levelof protective immunity directed to the tumour cell (or neoplasticcondition), pathogen or parasite, the clinical outcome in the patientsuffering from such a neoplastic condition or infection with a parasiteor a pathogen can be improved by also treating the patient with asuitable chemotherapeutic agent, as known to the art. Where the pathogenis viral, an anti-viral compound such as acyclovir can be administeredconcomitantly with the lipid binding polypeptide/antigen particlevaccination in patients with herpes virus infection, or HAART (highlyactive anti-retroviral therapy) in individuals infected with HIV. Wherethe pathogen is a bacterial pathogen, an antibiotic to which thatbacterium is susceptible is desirably administered and where thepathogen is a fungus, a suitable antifungal antibiotic is desirablyadministered.

Similarly, chemical agents for the control and/or eradication ofparasitic infections are known and are advantageously administered tothe human or animal patients using dosages and schedules well known tothe art. Where the patient is suffering from a neoplastic condition, forexample, a cancer, the administration of the immunogenic compositioncomprising the lipid binding polypeptide/antigen particles carrying oneor more multiplicity of cancer-associated antigens in the patient towhich it has been administered is desirably accompanied byadministration of antineoplastic agent(s), including, but not limitedto, such chemotherapeutic agents as daunorubicin, taxol, thioureas,cancer-specific antibodies linked with therapeutic radionuclides, withthe proviso that the agent(s) do not ablate the ability of the patientto generate an immune response to the administered lipid bindingpolypeptide/antigen particles and the antigens whose expression theydirect in the patient. Nucleic acids for modulating gene expression orfor directing expression of a functional protein can be incorporatedwithin the particles, especially where the nucleic acid molecules formcomplexes with cationic lipids, many of which are commerciallyavailable.

The virus or cell comprising the antigen molecule can be provided forinstance as a virus, or virus-like particle. The solubilizing agent forsolubilizing a structure comprising the antigen molecule can be asolvent such as HEGA-10, HEGA-11, MEGA-10, Cymal-5, or any other knownsolubilizing agent that has been proven to preserve an intact antigenmolecule such as a membrane protein upon solubilisation.

In addition to the hydrophobic membrane component, that serves asantigen, further lipid components can be used for the preparation of thelipid-binding polypeptide/antigen particles. They can be selected fromnaturally occurring lipids, synthetic lipids, modified lipids, fats,waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides,triglycerides, phospholipids, fatty acids, glycerolipids,glycerophospholipids, sphingolipids, saccharolipids, polyketides, sterollipids and prenol lipids or combinations thereof.

Pharmaceutical formulations, such as vaccines or other immunogeniccompositions, of the present invention comprise an immunogenic amount ofthe antigen-bearing particles in combination with a pharmaceuticallyacceptable carrier. An “immunogenic amount” is an amount of theantigen-bearing particles which is sufficient to evoke an immuneresponse in the subject to which the pharmaceutical formulation isadministered. Depending on the setting for administration (i.e., diseasetreatment or prevention), the dose (and repetition of administration)can be chosen to be therapeutically effective or prophylacticallyeffective.

Exemplary pharmaceutically acceptable carriers include, but are notlimited to, sterile pyrogen-free water and sterile pyrogen-freephysiological saline solution. Subjects which may be administeredimmunogenic amounts of the antigen-carrying particles of the inventioninclude, but are not limited to, human and animal (e.g., dog, cat,horse, pig, cow, goat, rabbit, donkey, mouse, hamster, monkey) subjects.Immunologically active compounds such as cytokines and/or BCG can alsobe added to increase the immune response to the administered immunogenicpreparation.

Immunogenic compositions comprising the lipid bindingpolypeptide/antigen particles which incorporate antigens of interestproduced using the methods of the invention may be formulated by any ofthe means known in the art. Such compositions, especially vaccines, aretypically prepared as injectables, either as liquid solutions orsuspensions. Solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The active immunogenicingredients are advantageously mixed with excipients or carriers thatare pharmaceutically acceptable and compatible with the activeingredient. Suitable excipients include but are not limited to sterilewater, saline, dextrose, glycerol, ethanol, or the like and combinationsthereof.

In addition, if desired, the immunogenic compositions, includingvaccines, may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, and/or adjuvantswhich enhance the effectiveness of the vaccine. Examples of adjuvantswhich may be effective include but are not limited to aluminiumhydroxide; N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r-2′-dipalmitoyl-sn-glycero-3hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE); and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. The effectiveness of an adjuvant may be determined bymeasuring the amount of antibodies directed against the immunogeniccomponent of the nanoscale particles after administration. Suchadditional formulations and modes of administration as known in the artmay also be used.

The immunogenic (or otherwise biologically active) antigen-containingparticle compositions can be administered in a manner compatible withthe dosage formulation, and in such amount as will be prophylacticallyand/or therapeutically effective. Precise amounts of the activeingredient required to be administered may depend on the judgment of thephysician, veterinarian or other health practitioner and may be peculiarto each individual, but such a determination is within the skill of sucha practitioner.

The vaccine or other immunogenic composition may be given in a singledose or multiple dose schedules. A multiple dose schedule is one inwhich a primary course of vaccination may include 1 to 10 or moreseparate doses, followed by other doses administered at subsequent timeintervals as required to maintain and or reinforce the immune response,e.g., at weekly, monthly or 1 to 4 months for a second dose, and ifneeded, a subsequent dose(s) after several months or years. Hydrophobicor partially hydrophobic antigens can be incorporated into the particlesas described for other molecules, such as membrane proteins or smallmolecules. Where the antigen is in nature associated with or is within amembrane, either a solubilized pure or partially pure preparation or asolubilized membrane or membrane fragment preparation can be used as thesource of the input antigen in the particle assembly mixture.

According to an embodiment of the invention, the antigen molecule isreconstituted in a lipid-binding polypeptide that comprises anamphipathic a-helical peptide having a hydrophobic or neutral face and ahydrophilic face.

According to an embodiment of the invention an antigen moleculecomprising an integral membrane protein is solubilized andreconstituted.

Herein this disclosure, the term “integral protein particle” includesany of an integral membrane protein, integral membrane protein complex,a peripheral membrane protein and peripheral membrane protein complexwithout any limitation.

According to an embodiment of the invention, the reconstituted integralmembrane protein particle is an HIV spike protein.

In this way, a dynamical and flexible HIV spike protein can be correctlypresented to an individual's immune system during vaccination, which hasnot been presented until now according to our best knowledge despite theextensive research efforts being made under a long period of time.

The uncomplicated method according to the invention is suitable forindustrial large scale production of reconstituted membrane proteinssuitable for vaccine production or tools for vaccine production, inparticular for vaccine production of vaccines against viral fusionproteins of type 1, type 2, type 3, such as HIV vaccines, in particularHIV-1 vaccines. But, the method according to the invention is alsosuitable for other membrane enveloped viruses than viral fusion proteinsincluding other integral membrane proteins, than the above HIV spikeprotein, such as other integral membrane protein complexes frombacteria.

Alternatively, the method according to the invention may also be appliedto integral membrane proteins from eukaryote cells or other peripheralmembrane proteins.

Herein, the term “peripheral membrane protein” is referred to protein orprotein complexes that are only temporarily associated to lipidmembranes. Typically, peripheral membrane proteins exist in viruses,bacteria and eukaryotes.

According to an embodiment of the invention, there is provided animmunogenic composition comprising lipid-binding polypeptide/antigenparticles comprising at least one hydrophobic or partially hydrophobicantigen molecule from a virus, a bacterium, fungus, protozoan, parasite,a human neoplastic cell or an animal neoplastic, tumour or cancer cell,wherein said immunogenic composition optionally further comprises aknown immunological adjuvant.

According to another embodiment of the invention there is provided areconstituted antigen particle comprising at least one hydrophobic orpartially hydrophobic antigen molecule from a virus, a bacterium,fungus, protozoan, parasite, a human neoplastic cell or an animalneoplastic, tumour or cancer cell, reconstituted in a lipid membranemimicking environment selected from a lipid-binding polypeptide suchthat the antigen molecules(s) is/are assembled therein.

Preferably the lipid-binding polypeptide is a saposin-like protein.According to a further preferred embodiment the saposin-like protein isselected from one or more of Saposin A, B, C and D. Particularlypreferred is saposin A.

According to yet another aspect of the invention there is provided a useof an antigen, such as a reconstituted membrane protein as a tool forvaccine production, for instance a tool to find and characterize properHIV immune response, i.e. use them as diagnostic tools to measure theproper antibody response in HIV infected patients or follow the sameresponse for vaccinated experimental animals or vaccinated patients.

According to another aspect of the invention, there is provided aprophylactic or therapeutic use of an immunogenic composition,comprising the lipid binding polypeptide/antigen particle describedherein.

BRIEF DESCRIPTION OF DRAWING FIGURES

To further explain the invention, embodiments and examples thereof theinvention will now be described in greater detail with reference to thedrawings.

FIG. 1 is a schematic illustration of a retrovirus such as a HIV-1virus;

FIG. 2 is a schematic illustration of a method for reconstituting amembrane protein according to an embodiment of the invention, inparticular for reconstituting spike protein particles for Salipro(Saposin A)-spike protein particle production;

FIG. 3 schematically illustrates the solubilisation of radioactivelylabelled HIV-1 VLP with high CMC detergents.

FIG. 4 shows the optimisation of the amount of Saposin A needed forefficient Salipro-HIV-spike nanoparticle formation.

FIG. 5 shows BN-PAGE and silver stain analyses of Saposin A-POT1 andpurified Saposin A-HIV-spike particles;

FIG. 6 illustrates stability, preserved structure and functionality ofHIV-1 spike in Saposin A nanoparticles;

FIG. 7 illustrates negative stain EM analysis of purified SaposinA-HIV-spike particles.

FIG. 1 is a schematic illustration of a retrovirus particle. The virionis a spherical particle surrounded by a phospholipid bilayer whichharbours the viral spike proteins. A HIV-1 virus particle typicallycomprises a viral lipid membrane enveloping a matrix layer, whichenvelopes viral RNA, Capsid, Integrase and Reverse Transcriptase. Thelipid membrane is integrated with HIV spike proteins being anchored atmultiple positions. The HIV spike protein can be considered to be anintegrated membrane protein. In HIV virus particles, the proteinsexpressed in the viral lipid membrane are called “Envelopeglycoproteins” (Env). Env contains two subunits that are non-covalentconnected, referred to as gp120-gp41. The HIV spike protein is anoligomer, being a trimer of a gp120/gp41 subunit pair of a docketingglycoprotein/trans-membrane glycoprotein complex, anchored in the virallipid membrane 2 and needed for the virus to enter target cells (notillustrated). Thus, the trimers of gp120/gp41-sub-unitpairs can beconsidered to form the spike proteins (Adapted from Retroviruses, ColdSpring Harbour Press, J. M. Coffin, S. H. Hughes and H. E. Varmous,1995).

It has been realized that isolated bnAb targets the spike proteins andconsequently a working vaccine should contain this protein complex.Further it has been realized that a complete functional spike protein asit appears in the virus membrane would constitute an ideal antigen,since the spike proteins are the only proteins on intact HIV-1particles. Thus, an immune response against the spike proteins isimportant to stop virus particles to enter host cells. This differs fromand is an advantage to other vaccine strategies which use HIV proteins,but will not prevent virus particles to spread. A problem is that spikeproteins are unstable complexes and it's not known until how to preservea functional spike protein, which is considered to be essential forbeing able to produce a functional vaccine against HIV. It has beenrealized that even HIV (virus) particles may contain incompleteassembled or disassembles spike proteins.

FIG. 2 is a schematic illustration of a method for reconstituting amembrane protein according to an embodiment of the present invention, inparticular for reconstituting spike protein particles for saposin-spikeprotein particle production.

FIG. 2(a) shows a schematic illustration of the HIV-1 spike protein inthe viral membrane. The HIV-1 Envelope glycoprotein, i.e. the spike,consists of two subunits, the peripheral gp120 subunit (brown) and thetransmembrane gp41 subunit (grey), forming a hetrotrimer. The bindingsite for the CD4 receptor and epitopes for the antibodies PG16 and 17bare shown (dark brown). Note that the 17b epitope is hidden in thenative spike structure, but becomes exposed after CD4 receptor binding.

FIG. 2(b) shows a schematic illustration of the saposin-HIV-spikereconstitution. Purified VLPs containing HIV-1 spikes were mixed withsaposin A and solubilized with HEGA-10 followed by detergent removalusing a desalt spin column. The removal of the detergent allows theformation of saposin-HIV-spikes.

FIG. 2(c) illustrates the reconstitution of the HIV-1 spike protein intosaposin/antigen nanoparticles. BN-PAGE analysis resolves theradioactively labelled trimeric HIV-1 spike protein (gp3) after VLPsolubilisation using HEGA-10 (lane 1). A fraction of dissociatedglycoprotein (gp) monomers were also observed. If HEGA-10 is removed,using a desalt column, the HIV-1 spike proteins aggregate and cannot beresolved by BN-PAGE (lane 2). Importantly, when saposin A was presentduring HEGA-10 removal the trimeric spike protein was resolved by theBN-PAGE, suggesting reconstitution of the HIV spike into saposin/antigennanoparticles (lane 3). Shown is a phosphor image of the gel.

In FIG. 2(d) the purification of saposin-HIV-spike complexes is shown.Unlabelled VLPs from 14 transfected cell culture flasks (150 cm²) wereused for reconstituting saposin-HIV-spike particles as described. Theparticles were affinity purified by Galanthis Nivalis chromatograpy.Shown are non-reducing SDS-PAGE analyses of the purified VLP preparation(lane 1), the crude reconstituted saposin/antigen particle preparation(lane 2) and the lectin purified saposin/antigen preparation (lane 3).The migration of the disulfide linked gp120-gp41 complex, the capsid(CA) and the matrix (MA) proteins of the virus as well as the saposin Aare indicated. The gel was stained for protein using Sypro Ruby.

FIG. 2(e) illustrates that the HIV-1 spike is stable, has a native foldand preserves its function in saposin-HIV-spike nanoparticles.Radioactively labelled and lectin purified saposin-HIV-spike particleswere incubated at 37° C. for 16 h followed by 2 h incubation at 37° C.with (lanes 3-6) or without (lane 2) 10 μg/ml of the HIV-1 spikeligands, PG16 Ab (150 KDa) (lane 3), sCD4 (50 KDa) (lane 4), sCD4 and17b Fab (50 KDa) together (lane 5) or 17b Fab alone (lane 6), andanalyzed by BN-PAGE. A control sample was kept on ice without ligands(lane 1). Binding of the ligands were followed by the shift in bandmigration of the saposin-HIV-spike nanoparticle complexes. Note thatonly one PG16 Ab can bind to the trimeric spike protein due to stericreason. 17b Fab and sCD4 bind stochiometrically, adding about 150 KDaeach in molecular weight to the complex, but the latter complex movesslower in the gel. Shown is a phosphor image of the gel.

FIG. 3 schematically illustrates the solubilisation of radioactivelylabelled HIV-1 VLP with high CMC detergents. HIV-1 VLPs were solubilisedin 1× HNC buffer containing 25 mM Anameg-7 (CMC 19.5 mM) (lane 2), 9 mMHEGA-10 (CMC 7 mM) (lane 3), 14 mM C-HEGA-11 (CMC 11.5 mM) (lane 4), 9mM MEGA-10 (CMC 6-7 mM) (lane 5), 12 mM n18Octyl-beta-D-Thiomaltopyranoside (OT) (CMC 9 mM) (lane 6), or 10 mMTetraethylene Glycol Monooctyl Ether (C8E4) (CMC 10 mM) (lane 7), for 10min on ice (a) or for 30 min at 37° C. (b) and analysed by BN-PAGE. VLPsolubilised in TX100 (TX) on ice was analyzed as control (lane 1).Migration of spikes and gp monomers are indicated. Note the dissociationof spikes into monomers by the 37° C. incubation.

FIG. 4 shows the optimisation of the amount of saposin A needed forefficient saposin-HIV-spike nanoparticle formation. Radioactivelylabelled VLPs were mixed with saposin A (230-0.77 μg/ml) followed by 10min solubilisation on ice using 9 mM HEGA-10 in 1× HNC buffer. HEGA-10was then removed and the amount of reconstituted saposin-HIV-spikeparticles was monitored by BN-PAGE. About 100 μg/ml saposin A was foundto be optimal.

The saposin/antigen particles used in the invention are a nanoparticlesystem of a saposin-like compound, which can oligomerize at properconditions forming a cluster of nanoparticles, instead of micelles,where an inside of the nanoparticles (of the cluster) provides a lipidmembrane mimicking environment.

FIG. 6 illustrates that the HIV-1 spike is stable, has a native fold andpreserves its function in saposin/antigen nanoparticles for at least 90h at 37° C. Radioactively labelled and lectin purified saposin-HIV-spikeparticles were incubated at 37° C. for 90 h followed by 2 h incubationat 37° C. with (lanes 3-6) or without (lane 2) 10 μg/ml of the HIV-1spike ligands, PG16 Ab (150 KDa) (lane 3), sCD4 (50 KDa) (lane 4), sCD4and 17b Fab (50 KDa) together (lane 5) or 17b Fab alone (lane 6), andanalyzed by BN-PAGE. A control sample was kept on ice without ligands(lane 1). Binding of the ligands were followed by the shift in bandmigration of the saposin-HIV-spike nanoparticle complexes, similar asfigure Xd. Note the preservation of the HIV-1 spike structure in thesaposin-HIV-spike particles after this extreme incubation.

In FIG. 7 a negative stain EM analysis of purified saposin-HIV-spikeparticles is provided. Shown is a raw EM image (left panel), and someselected particles (right panel). These are of similar size as earlierpublished HIV-1 spike structures.

The product of the process of the invention is present in a solution. Itcan be lyophilized or deep freezed.

The following examples further describe the invention.

EXAMPLE 1

Specifically, HIV-1 virus like particles (VLP) is produced by calciumphosphate-mediated DNA transfection of 293T cells. VLP released into thecell culture media are then purified by ultracentrifugation (BeckmanSW55 rotor, 28,000 rpm for 17 h) in a 20-60% sucrose density stepgradient. To generate saposin-spikes, the VLPs must first be solubilizedby a traditional detergent and then exchanged to saposin A. This ispossible to do by molecular sieving if the detergent has a high criticalmicelle concentration (CMC), i.e. in the mM range. Furthermore, thedetergent must be mild enough to preserve the native trimeric structureof the spike during solubilization. In these experiments we use thedetergent HEGA-10, with CMC at 7 mM. Accordingly, purified VLPs arelysed for 10 min on ice with 9 mM HEGA-10 in the presence of 90 μg/mlsaposin A. Then HEGA-10 is removed from the sample using a Zebadesalting spin column, 7 kDa cut off (Thermo Fisher Scientific)according to the instructions in the User Manual. The column onlyremoves the HEGA-10 detergent and not the saposin, which replaces thedetergent in the spike detergent complexes. The saposin-spikes productelutes from the column in the void volume. If saposin A was omitted fromthe experiment, the HIV-1 spikes aggregated and could not be resolved inBN-PAGE. FIG. 2 illustrates the method that was developed for fast andefficient saposin-spike production.

The particles obtained by the method of the invention according tovarious embodiments are robust over concentrating using standardcentrifugal filter units, freezing and thawing. Also, practicalexperiments revealed that the particles of the invention display acertain degree of thermo stability. In addition, it is probably possibleto freeze-dry, store and re-hydrate the particles of the inventionwithout any major quality deterioration observable. Experiments haveproved that HIV-spike protein particles are stable over a period of timeof over 90 hours at 37° C. with preserved structure and functionality,which proves long-term stability (FIG. 6). It has also been proved thatknown neutralising HIV-1 antibodies bind strongly to these spike proteinparticles and that even more importantly, that no neutralizingantibodies bind to the spike protein particles. This is exactly what isrequired for being able to produce an efficient HIV-1 vaccine. Thus, thespike protein particles are suitable for this application. They may alsobe suitable as development tools for vaccines.

Embodiments of the present invention are concerned with vaccinesprotective against HIV, in particular HIV-1 and with novel reconstitutedmembrane protein particles and a method for producing the same for usein such vaccines, vaccine compositions or tools for producing orassisting when producing the same, for instance kits for quickreconstitution of membrane enveloped proteins at the same time providingprotein stability as well as conserved structure and functionality, butis also applicable to other viruses such as flue, Ebola and SARS Theinvention is suitable for large-scale production.

EXAMPLE 2 Purification of Saposin A

Purified saposin A was prepared as follows. Saposin A protein expressionwas carried out using a vector with the coding region for human saposinA (SEQ ID NO: 1) inserted into a pNIC-Bsa4 plasmid and transformed andexpressed in E. coli Rosetta gami-2 (DE3) (Novagen) strains. Cells weregrown at 37° C. in TB medium supplemented with Tetracycline,Chloramphenicol and Kanamycin and induced with 0.7 mM IPTG. Three hoursafter induction, the cells were collected by centrifugation at 12.000×gfor 15 min. The supernatant was discarded, the cell pellet wasresuspended using lysis buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 20 mMImidazol) and disrupted by sonication. Lysates were subjected tocentrifugation at 26.000×g for 30 min, the supernatant heated to 85° C.for 10 min, followed by an additional centrifugation step at 26.000×gfor 30 min. Preparative IMAC purification was performed bybatch-adsorption of the supernatant by end-over-end rotation with NiSepharose™ 6 Fast Flow medium for 60 min. After binding of saposin A tothe IMAC resin, the chromatography medium was packed in a 10-mm-(i.d.)open gravity flow column and unbound proteins were removed by washingwith 15 bed volumes of lysis buffer. The resin was washed with 15 bedvolumes of wash buffer WB2 (20 mM Hepes pH 7.5, 150 mM NaCl, 40 mMImidazol). Saposin A was eluted by addition of five bed volumes ofelution buffer EB (20 mM Hepes pH 7.5, 150 mM NaCl, 400 mM Imidazol).The eluate was dialyzed overnight against gel filtration buffer GF pH7.5 (20 mM Hepes pH 7.5, 150 mM NaCl) supplemented with recombinant TEVprotease. TEV protease containing an un-cleavable His-tag was removedfrom the eluate by passing it over 2 ml IMAC resin. Cleaved targetproteins were concentrated to a volume of 5 ml using centrifugal filterunits and loaded onto a HiLoad Superdex™ 200 16/60 GL column using anAKTAexplorer™ 10 chromatography system (both GE Healthcare). Peakfractions were pooled and concentrated to 1.2 mg/ml protein. The proteinsample was flash frozen in liquid nitrogen and stored at −80 C.

1. A method for producing an antigen comprising at least one hydrophobicor partially hydrophobic antigen molecule from a virus, a bacterium,fungus, protozoan, parasite, a human neoplastic cell or an animalneoplastic, tumour or cancer cell, the method comprising the steps ofproviding a virus, or cell comprising an antigen molecule, purifying thecell comprising the antigen molecule, solubilizing the antigen moleculein a solubilizing agent that preserves intact an antigen molecule uponsolubilisation, reconstituting the antigen molecule in a lipid-bindingpolypeptide that provides a lipid membrane mimicking environment,wherein the lipid binding protein is a saposin-like protein.
 2. Themethod according to claim 1, wherein a virus or cell comprising anantigen molecule is provided.
 3. The method according to claim 2,wherein the antigen molecule is an integral membrane protein, anintegral membrane protein complex, a peripheral membrane protein or aperipheral membrane protein complex.
 4. The method according to claim 3,wherein the integral membrane particle is a viral spike protein, such asa trimeric envelope glycoprotein spike of HIV-1.
 5. (canceled)
 6. Themethod according to claim 1, wherein the saposin-like protein isselected from saposin A, saposin B, saposin C and/or saposin D.
 7. Themethod according to claim 1, wherein the antigen molecule isreconstituted in a saposin-like protein without adding any additionallipids.
 8. The method according to claim 1, wherein the antigen moleculeis solubilized in a solubilizing agent selected from one or more ofHEGA-10, C-HEGA-11 or MEGA-10.
 9. A reconstituted antigen particlecomprising at least one hydrophobic or partially hydrophobic antigenmolecule from a virus, a bacterium, fungus, protozoan, parasite, a humanneoplastic cell or an animal neoplastic, tumour or cancer cell,reconstituted in a lipid membrane mimicking environment selected from alipid binding polypeptide such that the antigen molecules(s) is/areassembled therein, wherein the lipid binding protein is a saposin-likeprotein.
 10. The reconstituted antigen particle according to claim 9,wherein the lipid membrane mimicking environment is a saposin-likeprotein, and interacts with the antigen molecules assembling them intothe saposin-like protein particles.
 11. The reconstituted antigenparticle according to claim 10, wherein the saposin-like protein isselected from one or more of saposin A, saposin B, saposin C and saposinD.
 12. The reconstituted antigen particle according to claim 8, whereinthe lipid membrane mimicking environment is a saposin-like protein andwherein the antigen molecules are HIV-1 spike proteins.
 13. Animmunogenic composition comprising the reconstituted antigen particle ofanyone of claims 9 to
 12. 14. Use of the immunogenic composition ofclaim 13 for vaccine production.
 15. The immunogenic composition ofclaim 13 for prophylactic or therapeutic vaccination.